APPLICANT'S ABSTRACT: We hypothesize that ethanol-induced chronic pancreatitis is triggered by hypoxia/reoxygenation injury, leading to stimulation of inflammatory cell recruitment and activation of these cells by circulating endotoxins. We recently showed pancreatic injury characteristic of chronic pancreatitis in humans in an enteral model modified to deliver higher amounts of ethanol. Our first goal is to characterize this new model. Wistar rats will receive alcohol internally using our modified protocol for up to 6 months. Pancreatic function and injury will be determined and compared to changes in key molecular events. We expect that alcohol will cause progressive, irreversible changes in pancreatic morphology and function, similar to clinical observations. We will next test the hypothesis that alcohol causes hypoxia in pancreas in vivo. Initially, we will determine if the hypoxia marker pimonidazole can be used to index pancreatic hypoxia in rats. We will then determine the effect of alcohol on pancreatic hypoxia in our enteral model. We expect that chronic ethanol will cause early increases in hypoxia, suggesting that hypoxia might play a key early role in the initiation of damage. The effect of removing endotoxin by lactobacillus treatment to displace Gram-negative bacteria, or killing macrophages with silica will next be studied. We expect pancreatic damage will be blunted by these treatments, suggesting a role of endotoxin and macrophages in chronic alcoholic pancreatitis. Next, the hypothesis that free radicals play a causative role in chronic alcoholic pancreatitis will be tested. This question will be addressed using adeno-associated virus (RAAV) to deliver superoxide dismutase (SOD) and cause stable long-term pancreatic expression. First, optimal conditions to confer pancreatic transgene expression will be determined. The effect of RAAV containing superoxide dismutase (SOD) on pancreatic injury will be determined. Free radical formation and oxidative stress will also be quantitated. We expect that preventing oxidative stress with SOD will protect against chronic alcoholic pancreatitis, indicating that free radicals play an important role in the progression of chronic alcoholic pancreatitis. This work will lay the mechanistic foundation for future studies of targeted therapies to prevent alcoholic pancreatitis that can applied in the clinic, where such therapy is desperately needed. Further, through didactic training and interactions with his mentor and key faculty in the enteral model of alcohol exposure, molecular biology, and viral vector gene therapy, the applicant will acquire new skills that will greatly enhance his career development and bridge the gap to becoming a successful member of the academic and scientific community.